My suggestion is ,don't take it lightly. Use both isotype controls.

Best regards, 

Babu

Hi Sergio

First, IgG3 and IgG1 not only differ in the hinge region but also in the Fc part. IgG3 activate the classical complement pathway a bit better than IgG1 but opsonisation is a bit more efficient with IgG1. It also seems that IgG3 can aggregate through their Fc region, although I am not 100% of this.

Anyway, there is no real answer to your question. You have to know that isotype controls antibodies are qualitative controls for so called “non-specific” binding (there could be a whole debate on that point), not qualitative references. In other words you cannot set a positivity threshold based on an isotype control signal intensity.

You are obviously very concerned by the rigor of your science, isotype controls can only disappoint you ! Here is my favourite quote from the Clinical and Laboratory Standards Institute guideline H42-A2:

« An isotype control antibody will never be entirely representative for the particular antibody for which it should control »

Kind regards

There is quite a lot of debate about the use of isotypic controls. I suggest you to read the article published in Cytometry A back in 2006 by Holden T. Maecker Flow cytometry controls, instrument setup, and the determination of positivity" 10.1002/cyto.a.20333 

Thank you very much! And for doing a high througput in 96-well plates there is any requeriment? The goal is the characterization of cell surface markers from non-human MSCs.

Sergio

I have limited experience with HTS, but I think it all depends on what you are looking for.  I suggest that you take a look in The article published by Erroll Strain et al in Advances in Bioinformatics in 2009 (Article ID 356141,  doi:10.1155/2009/356141).  

I will agree with Gulderen, Isotype control is not the right control. Read the recent article on debate on isotype control as well as the work handbook of BD, where they have clearly shown how isotype control can lead you to different results.